Breast Ductal Carcinoma Cells Research Articles

Effects of pargyline on cellular proliferation in human breast cancer cells

Monoamine oxidase (MAO) inhibitors have been tested for the purpose of treating many types of cancers including breast cancer. Pargyline, an MAO inhibitor, prevents the activity of lysine-specific demethylase 1 (LSD1), which is excessively expressed in breast cancer. The purpose of this study is to investigate the effect of pargyline on cellular proliferation in human breast ductal carcinoma (T47D) cells. After exposing T47D cells to pargyline, we examined cell proliferation, cell cycle, apoptosis, and the expression levels of apoptosis-related proteins and LSD1. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner. The treatment of 2 mM pargyline exhibited an increase of the cell proportion at the G1 phase, while the major decrease of the cell proportion was at the S phase compared to the controls. In addition, pargyline induced an increase in the cleaved PARP expression. However, we did not find a change in the LSD1 expression in the cells exposed to pargyline. Based on our results, it is believed that pargyline has pharmaceutical potential as an anticancer drug for the treatment of human breast cancer.

Human Adipose Tissue Macrophages Display Activation of Cancer-related Pathways

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Immunotoxin targeting CD133(+) breast carcinoma cells.

CD133 expression enriches for tumor-initiating cells and is a negative prognostic factor in numerous cancers. We previously developed an immunotoxin against CD133 by fusing a gene fragment encoding the scFv portion of an anti-CD133 antibody to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, demonstrated potent antitumor activity following intratumoral delivery into head neck cell carcinoma xenografts. However, the efficacy against other tumors and the tolerability of systemic administration remained unclear. The purpose of this study was to evaluate the tolerability and efficacy of dCD133KDEL in a systemic human breast carcinoma model. Time course viability studies showed that dCD133KDEL selectively inhibited MDA-MB-231 ductal breast carcinoma cells that contained a minority CD133(+) subpopulation, implicating CD133(+) cells as a source for self-renewal within this cell line. Furthermore, systemic administration of dCD133KDEL caused regression or inhibition of tumor growth in mice bearing an intrasplenic MDA-MB-231 tumor challenge as a model for metastatic disease. In the same model, combined therapy with dCD133KDEL and another immunotoxin designed to target the bulk tumor mass was the most effective therapy, supporting the idea that such combination therapies might better address tumor heterogeneity. dCD133KDEL shows promise as a therapeutic agent and as a biologic tool to study cancer stem cells.

Abstract 1176: Bypassing the dominant-negative effect of mutant p53 in cancer cells

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. The transcriptional activity of p53 depends on localization to the nucleus and formation of p53 tetramers. Use of wild-type (wt) p53 for gene therapy is limited due to the dominant-negative effect of mutant (mut) p53 over wt p53. Mut p53 will hetero-oligomerize with wt p53 via the tetramerization domain (TD) found in p53, deactivating the tumor suppressor function of wt p53. In order to use p53 for gene therapy, an alternative oligomerization domain for p53 was investigated. The coiled-coil (CC) domain from Bcr (breakpoint cluster region), similar to the native TD of p53, can form an antiparallel dimer of dimers. The TD from p53 was then substituted with this CC, and the activity of this new p53 (p53-CC) was tested. We evaluated the transcriptional activity of p53-CC compared to wt p53 in a dual luciferase reporter gene assay. To demonstrate the ability of our p53-CC to bypass the dominant-negative effect prompted by hetero-oligomers, the reporter gene assay was carried in human breast adenocarcinoma (MCF-7) and human breast ductal carcinoma (T47D) cell lines. MCF-7 cells contain wt p53 population that is mislocalized to the cytoplasm, whereas, T47D cells contain mutant p53. Indeed, data from the reporter gene experiment shows a reduction in wt p53 activity introduced to T47D cells compared to MCF-7 cells, while the activity of p53-CC remains consistent regardless of the presence of an endogenous mut p53 population. We also tested the biological activity of p53-CC and its ability to induce apoptosis using 7-AAD and Annexin-V assays (late stage apoptosis). In addition, Caspase-3/7 assay (mid-stage apoptosis) was performed to further validate a p53-dependent apoptotic pathway. Data obtained from the aforementioned experiments suggest that p53-CC has a very similar biological activity to wt p53. Furthermore, we examined the gene expression profile of p53-CC using wt p53 as a reference. The assay was performed using real-time PCR array technology to analyze the expression of 84 genes related to p53-mediated signal transduction. Two negative controls were used; the first is a p53 construct that lacks its tetramerization domain (p53ΔTDC) and should not possess any transcriptional activity, and the second is the CC by itself. Indeed, p53-CC shows comparable gene expression profile to wt p53 and has the ability to transactivate the various p53 target genes. In summary, p53-CC retains p53 activity and excludes hetero-oligomer formation, overcoming a major barrier in using p53 for cancer gene therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1176. doi:1538-7445.AM2012-1176

Investigation of cytotoxic activity in four stachys species from iran.

The aerial parts of Stachys laxa Boiss. and Buhse. from Siah-bishe in Mazandaran province, Stachys trinervis Aitch. and Hemsl. from Karaj in Alborz province, Stachys subaphylla Rech. F. and Stachys turcomanica Trautv. from Golestan province have been collected in May 2008. Total extracts were obtained through MeOH/H2O (80/20) and then partitioned between CHCl3, EtOAc and MeOH. These fractions and total extracts have been investigated for in-vitro cytotoxic activity against the colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D) and Swiss mouse embryo fibroblast (NIH 3T3) cell lines using MTT assay (3-(4,5-di methyl thiazol-2-yl)-2,5-di phenyltetrazolium bromide). At each cell line, doses of 3.125, 6.25, 12.5, 25, 100, 200, 400 and 800 µg/mL in 1% (v/v) DMSO of all samples were tested. Ethyl acetate and chloroform fractions of Stachys laxa against proliferation of T47D and HT-29 cell lines and chloroform fraction of Stachys subaphylla and Stachys subaphylla ethyl acetate fraction toward T47D cell line exhibited highest cytotoxic activity (IC50 < 50 µg/mL). Ethyl acetate and chloroform fractions of Stachys turcomanica against HT-29 cell line, except methanol fraction of Stachys subaphylla, the other extrcts on T47D cell line, represented moderate cytotoxic activity (IC50 < 70 µg/mL). All fractions of S. trinervis demonstrated no effective cytotoxic activity. IC50 values confirmed that the growth and proliferation of HT-29 and T47D cells were most affected by chloroform and ethyl acetate fractions of Stachys laxa and Stachys turcomanica due to their nonpolar compounds.

Breast Cancer Subtype-Specific Interactions with the Microenvironment Dictate Mechanisms of Invasion

Most ductal breast carcinoma cells are weakly invasive in vitro and in vivo, suggesting that components of their microenvironment may facilitate a transition from in situ to invasive stages during progression. Here, we report that coculture of mammary fibroblasts specifically triggers invasive behavior in basal-type breast cancer cells through a ligand independent mechanism. When cultured alone in organotypic culture, both basal- and luminal-type breast cancer cells formed noninvasive spheroids with characteristics of ductal carcinoma in situ (DCIS). In contrast, when cocultured with mammary fibroblasts, basal-type spheroids exhibited invasive character whereas the luminal-type spheroids retained a benign and noninvasive duct-like architecture. Real-time imaging and functional studies revealed that the specificity of invasion was linked to a unique capacity of basal-type breast cancer cells to move within spheroids. Mammary fibroblasts induced invasion by triggering basal-type breast cancer cells to convert from a noninvasive program of mammary epithelial morphogenesis to an invasive program of sprouting endothelial angiogenesis. Contrary to the existing invasion models, soluble ligands produced by the fibroblasts were not sufficient to trigger invasion. Instead, basal-type invasion relied upon a Cdc42-dependent reorganization of collagen fibers in the extracellular matrix by fibroblasts. Inhibiting basal-type cell movement with clinically relevant drugs blocked invasion both in organotypic culture and in animals, suggesting a new treatment strategy for early-stage patients. Together our findings establish that fibroblast recruitment by basal-type breast cancer cells into early-stage tumors is sufficient to trigger their conversion from a benign, noninvasive DCIS-like stage to a malignant invasive stage. Furthermore, our findings suggest that different subtypes of breast cancer may require distinct types of contributions from the microenvironment to undergo malignant progression.

Epidermal growth factor‐dependent enhancement of invasiveness of squamous cell carcinoma of the breast

Factors that promote the aggressiveness of squamous cell carcinoma of the breast are not well understood. To examine the involvement of cell motility and the mechanism of this behavior, a squamous cell carcinoma cell line of the breast (HBC9) was established from a metastatic lymph node of a Japanese woman. HBC9 expressed epidermal growth factor receptor (EGFR), but was negative for Her2 or Her3.The invasive ability of HBC9 was compared with that of four breast ductal carcinoma cell lines by Matrigel invasion assay. EGF stimulation induced the formation of surface protrusions and cell migration in HBC9 cells, and significantly increased the number of cells migrating through the Matrigel. The invasive ability of HBC9 was compared with other cell lines of breast carcinoma; it was much greater than that of MCF-7, BT474, or HBC5, but did not differ significantly from that of MDA-MB-231. Observation of the surface protrusions of HBC9 by confocal laser microscopy revealed co-localization of Arp2 and N-WASP with actin polymerization, detected by visualization with phalloidin, indicating that the protrusions induced by EGF were invadopodia. In HBC9 cells, cortactin also co-localized with the N-WASP/Arp2/3 complex in the protrusions. Immunohistochemistry of 12 cases of squamous cell carcinoma of the breast revealed expression of cortactin and EGFR in all of them, and this was confirmed by western blotting in two cases. These results suggest that EGF-dependent enhancement of cell motility by formation of invadopodia associated with cortactin is a cause of the clinical aggressiveness of squamous cell carcinoma of the breast.

Abstract 3637: Sensitivity of breast cancer cell lines with distinct genotypes to poly(ADP-ribose) polymerase inhibitor or in combination with DNA damaging agents

Abstract Poly(ADP-ribose) polymerase (PARP) is required for the repair of single-strand DNA nicks and BRCA1/2 proteins are directly involved in the repair of DNA double-strand breaks through homologous recombination (HR). Recent studies have demonstrated that PARP inhibitors administered as single agents are active in 33 to 38% of BRCA-deficient advanced breast and ovarian cancer patients. However, the resistant mechanisms for the two thirds of patients with genetically-defined BRCA deficiency who did not respond to anti-PARP agents are largely undefined. The aim of this study was to test sensitivity of breast cancer cell lines with distinct genotypes to a PARP inhibitor alone or in combination with DNA damaging agents (topotecan and carboplatin). Methods: HCC1428 (BRCA2-mutant [2135basepair.del], but HR-competent breast adenocarcinoma); MDA-MB-231 (triple [ER, PR and HER2]-negative, BRCA-wildtype breast adenocarcinoma); and MCF-7 (BRCA-wildtype breast ductal carcinoma) cells were treated with carboplatin (1 μM), topotecan (10 nM), or veliparib (1 μM; ABT-888) alone or in combinations consisting of veliparib with topotecan or carboplatin for 24h. The survival of cancer cells was evaluated by clonogenic assays. Differences in cell survival (colony numbers) between vehicle-treated and veliparib-, DNA damaging agent-, or combination-treated were evaluated with unpaired t-tests and P values are two-sided. Results: Veliparib plus topotecan compared to topotecan alone markedly reduced the survival of MDA-MB-231 (mean ± SD; 36 ± 3 vs. 141 ± 4; P ≤ 0.01), MCF-7 (6 ± 1 vs. 25 ± 0.8; P ≤ 0.01), HCC1428 (10 ± 2 vs.29 ± 6; P = 0.04). The combination of veliparib and carboplatin versus carboplatin alone also reduced the survival of MDA-MB-231 (133 ± 2 vs. 477 ± 138; P ≤ 0.01), MCF-7 (48 ± 5 vs. 60 ± 2; P = 0.07) and HCC1428 (184 ± 6 vs. 260 ± 14; P ≤ 0.01). Veliparib has single agent activity in MCF-7 (53 ± 8 vs. 80 ± 4; P = 0.02) but not in BRCA2-mutant HCC1428 (259 ± 10 vs. 257 ± 13; P = 0.83) or triple-negative MDA-MB-231 (121 ± 2 vs. 104 ± 6; P = 0.54) cells. Conclusions: Veliparib enhances breast cancer cell death induced by DNA damaging agents. It has single agent activity in BRCA-wildtype MCF-7 cells, suggestive of other mechanisms of response to PARP inhibition in addition to BRCA deficiency. The BRCA2-mutant line HCC1428 is resistant to veliparib or carboplatin treatment alone but sensitive to topotecan alone or in combination with veliparib. The latter may provide an alternative for the management of PARP inhibitor- or platinum-resistant BRCA-mutant tumors. The underlying mechanisms of sensitivity or resistance to the PARP inhibitor alone or to the PARP inhibitor plus DNA damaging agents are under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3637.

A novel tumor suppressor gene RhoBTB2 (DBC2): Frequent loss of expression in sporadic breast cancer

RhoBTB2 was isolated recently as a tumor suppressor gene from human chromosome 8p21.3. Although RhoBTB2 was found to be frequently lost in breast cancer lines, expression status of RhoBTB2 in sporadic breast cancer tissues and its clinical and prognostic value, however, remain unclear. Tissue samples from breast cancer patients and normal controls and cell samples from cell lines were collected and reverse transcription (RT)-PCR was used to monitor the presence of RhoBTB2 mRNA. The protein expression of RhoBTB2 was detected by immunohistochemical staining. Cumulative survival time was assessed by the Kaplan-Meier method and Cox regression model. We discovered that RhoBTB2 expression was lacking in a breast ductal epithelial carcinoma cell line T-47D but was expressed in other types of tumor cell lines and normal tissues we tested. The results from tissue samples showed that RhoBTB2 was absent in 60% of breast cancers on both the mRNA and protein level. The results from RT-PCR were completely uniform with those from immunohistochemistry. We demonstrated that loss of RhoBTB2 more frequently occurred in postmenopausal patients of age >or=50 yr old and in patients with infiltrating ductal carcinoma of the breast. The prognostic value of RhoBTB2 in breast cancers also be assessed by a long-term follow-up investigation and we found that patients with RhoBTB2-negative breast cancer were linked to poor clinical prognosis. Therefore, the loss of RhoBTB2 expression is a common occurrence in breast cancers and it is an important factor in the development and prognosis of sporadic breast cancer.

On-chip testing device for electrochemotherapeutic effects on human breast cells

A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator. As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment.

Transduction of E2F-1 TAT fusion proteins represses expression of hTERT in primary ductal breast carcinoma cell lines

BackgroundTelomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.MethodsProtein purification techniques were refined to yield biologically active Tat fusion proteins (TFPs) capable of transducing the breast cancer cell lines HCC1937 and HCC1599. Cell lines were treated with wildtype E2F-1 (E2F-1/TatHA), mutant E2F-1 (E132/TatHA) and a control Tat peptide (TatHA) for 24 hours. Total RNA was isolated from treated cells, reverse transcribed and fold changes in gene expression for hTERT determined via real-time RT-qPCR.ResultsSignificant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).ConclusionThese results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.

Identification of BAF57 mutations in human breast cancer cell lines

Accumulating genetic and biochemical evidences support a role for the SWI/SNF chromatin-remodeling complex in cancer development and multiple core subunits of these complexes have been found to function as tumor suppressor genes. The core SWI/SNF subunit BAF57 mediates direct interactions with estrogen and androgen receptors (ER and AR) regulating their transcriptional activity. BAF57 gene maps to chromosome band 17 q21 in close proximity to the BRCA1 gene. This locus has been associated with frequent loss of heterozygosity (LOH) and allelic imbalance in breast cancers; however, BRCA1 mutations are rare events in sporadic breast cancer with LOH in the region, suggesting that another tumor suppressor gene resides in this area. All these reasons prompted us to screen for mutations in the BAF57 gene using a panel of the most commonly used human breast cancer cell lines. All cell lines analysed contain wild-type copies of BAF57 gene with the only exception of the breast ductal carcinoma cell line BT549. Sequencing of genomic DNA and cDNA generated from BT549 mRNA demonstrated the presence of a CA dinucleotide insertion in exon 5 of BAF57. The absence of wild-type BAF57 alleles indicates that this is a biallelic inactivating mutation that causes a frameshift and as a consequence a premature stop codon leading to a truncated BAF57 protein. A functional characterisation of the truncated BAF57 showed that it has lost the ability to bind to ER but still binds to the nuclear receptor coactivator SRC1e. Furthermore, we observed that the expression of the truncated BAF57 increased the ability of SRC1e to potentiate transcriptional activation by ERalpha, suggesting that mutations in BAF57 could contribute to the oncogenic transformation in breast cancer cells.

Monoclonal antibody 5C3 raised against formalin fixed paraffin-embedded invasive breast tumour tissue: Characterisation of its reactive antigen via immunoprecipitation and internal sequencing

Monoclonal antibodies (MAbs) provide a powerful tool for the identification of novel tumour associated antigens. In an attempt to identify such an antigen, MAbs were generated by immunization with paraffin wax-embedded formalin-fixed invasive ductal breast tumour tissue from a patient who relapsed following an initial response to adjuvant chemotherapy. Extensive immunocytochemical and Western blot analysis of a range of cell lines and tissues including a series of pre- and post-chemotherapy treated invasive ductal breast carcinomas, with one of these MAbs, antibody 5C3, indicated that the 5C3 reactive antigen displayed a wide spectrum of reactivity amongst various human tumours. A reduced level of 5C3 expression was observed in non-cancerous archival breast tissues and breast cell lines and normal murine tissues compared to the expression observed in infiltrating breast tumour cells. Immunoprecipitation studies using the human ductal breast carcinoma cell line, ZR-75-1 resulted in the isolation of a 175 kDa reactive band which was excised from an SDS-PAGE gel and subjected to internal sequencing. Sequencing analysis and database searching revealed that this 175 kDa band represented a cytokeratin heteropolymer, composed of type I cytokeratin 9 and type II cytokeratin 6. Further studies confirmed that antibody 5C3 recognised this heteropolymer of cytokeratin 9 and 6 but not the individual cytokeratins. This novel method of MAb generation may facilitate the isolation of further potentially interesting cellular antigens. Characterisation of these novel antigens may identify specific disease targets with possible prognostic or predictive significance.

Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line

Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours. This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. Human breast ductal carcinoma MCF7 cells were transfected using FuGENE 6 with 1 microg of pcDNA3-EGFR containing the full-length human EGFR promoter or 1 microg of the vectors alone (pcDNA3). The transfected cells were transferred into a 25-cm2 flask containing growth medium and G418. Confluent cultures were lysed, total protein levels measured and electrophoresed. The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4 degrees C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody. We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid. These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2. The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells. In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENE 6 Transfection Reagent. The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation.

Differential expression of high-affinity melatonin receptors (MT1) in normal and malignant human breast tissue.

Melatonin is a pineal hormone that strongly inhibits the growth of breast cancer cells in vitro and in vivo. We report thefirst use of immunohistochemical analysis to determine the distribution of the high-affinity melatonin receptor subtype, MTI, in human breast tissue, the hypothalamic suprachiasmatic nucleus, and skin. The MT1 antibody, which is specific for the cytoplasmic portion of the receptor, produced cytoplasmic staining in normal-appearing breast epithelial cells and ductal carcinoma cells; stromal cells, myoepithelial cells, and adipocytes were nonreactive. The majority of nonneoplastic samples (13/19 [68%]) were negative to weakly positive, while moderate to strong reactivity was seen in most cancer samples (49/65 [75%]). Thus, although MT1 receptors were detectable in normal and malignant breast epithelium, high receptor levels occurred more frequently in tumor cells (P < .001), and tumors with moderate or strong reactivity were more likely to be high nuclear grade (P < .045). These findings may have implications for the use of melatonin in breast cancer therapy.

PREOPERATIVE PLASMA LEVELS OF INTERLEUKIN-6 AND ITS SOLUBLE RECEPTOR PREDICT DISEASE RECURRENCE AND SURVIVAL OF PATIENTS WITH BLADDER CANCER

Elevated levels of interleukin-6 (IL-6) are associated with metastasis and poor prognosis in various malignancies. Since the IL-6 soluble receptor (IL-6sR) potentiates the systemic effects of IL-6, each may independently impact the disease process. We tested the hypothesis that preoperative plasma IL-6 and IL-6sR levels would predict cancer stage and prognosis in patients with transitional cell carcinoma of the bladder. The study group consisted of 51 patients who underwent radical cystectomy for transitional cell carcinoma and 44 men without cancer. Preoperative plasma levels of IL-6 and IL6sR were measured by enzyme-linked immunosorbent assay and correlated with pathological features and clinical outcome. IL-6 levels were higher in patients with bladder cancer than in healthy controls (p <0.001). In bladder cancer cases elevated levels of IL-6 and IL-6sR were associated with adverse pathological features, including muscle invasion, lymphovascular invasion and lymph node metastases (p <0.05). High levels of IL-6sR were also associated with pathological tumor grade (p = 0.036). In separate multivariate models that included clinical stage and grade IL-6 and IL-6sR levels were independent predictors of lymphovascular invasion, metastases to lymph nodes, disease recurrence and disease specific survival (p <0.05). In a preoperative Cox proportional hazards model IL-6 (p = 0.050) and IL-6sR (p = 0.035) predicted disease specific survival. We found that plasma IL-6 levels were higher in patients with bladder cancer than in healthy controls. Levels of IL-6 and IL-6sR were associated with cancer stage and metastases, and were strong independent predictors of disease recurrence and disease specific survival.

ONCOSTATIN M SUPPRESSES EGF-MEDIATED PROTEIN TYROSINE PHOSPHORYLATION IN BREAST CANCER CELLS

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.

Synthesis, in vitro pharmacologic characterization, and preclinical evaluation of N-[2-(1′-piperidinyl)ethyl]-3-[ 125I]iodo-4-methoxybenzamide (P[ 125I]MBA) for imaging breast cancer

The goal of this study was to investigate the potential use of a radioiodinated benzamide, N-[2-(1′-piperidinyl)ethyl]-3-iodo[ 125I]-4-methoxybenzamide (P[ 125I]MBA), a sigma receptor binding radioligand for imaging breast cancer. The chemical and radiochemical syntheses of PIMBA are described. The pharmacological evaluation of PIMBA was carried out for sigma-1 and sigma-2 receptor sites. The in vivo pharmacokinetics of the radioiodinated benzamide were determined in rats and comparison of P[ 125I]MBA with Tc-99m sestamibi were made in a rat mammary tumor model. Sigma-1 affinity (K i) for PIMBA in guinea pig brain membranes using [ 3H](+)pentazocine was found to be 11.82 ± 0.68 nM, whereas sigma-2 affinity in rat liver using [ 3H]DTG (1,3- o-di-tolylguanidine) was 206 ± 11 nM. Sites in guinea pig brain membranes labeled by P[ 125I]MBA showed high affinity for haloperidol, (+)-pentazocine, BD1008, and PIMBA ( K i =4.87±1.49,8.81±1.97,0.057±0.005,46.9±1.8 nM, respectively). Competition binding studies were carried out in human ductal breast carcinoma cells (T47D). A dose-dependent inhibition of specific binding was observed with several sigma ligands. K i values for the inhibition of P[ 125I]MBA binding in T47D cells for haloperidol, N-[2-(1′-piperidinyl)]ethyl]4-iodobenzamide (IPAB), N-( N-benzylpiperidin-4-yl)-4-iodobenzamide (4-IBP), and PIMBA were found to be 1.30 ± 0.07, 13 ± 1.5, 5.19 ± 2.3, 1.06 ± 0.5 nM, respectively. The in vitro binding data in guinea pig brain membranes and breast cancer cells confirmed binding to sigma sites. The saturation binding of P[ 125I]MBA in T47D cells as studied by Scatchard analysis showed saturable binding, with a K d =94±7 nM and a B max =2035±305 fmol/ mg of proteins. Biodistribution studies in Sprague–Dawley rats showed a rapid clearance of P[ 125I]MBA from the normal organs. The potential of PIMBA in imaging breast cancer was evaluated in Lewis rats bearing syngeneic RMT breast cancers, a cancer that closely mimics human breast cancer histology. At 1 h postinjection, tumor uptake for P[ 125I]MBA and Tc-99m sestamibi were found to be 0.35 ± 0.01 and 0.32 ± 0.01 % injected dose/organ (%ID/g), respectively. The %ID/g for liver, kidneys, and heart were 2, 11, and 20 times lower, respectively, for P[ 125I]MBA as compared with Tc-sestamibi. Slightly higher uptake of P[ 125I]MBA in tumors (than Tc-sestamibi) and a low nontarget organ uptake warrants further studies of this and other sigma receptor ligands for their use as breast cancer imaging agents.

Enhanced expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase gene in human breast tumor cells

The expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase ( 8-oxo-dGTPase) gene in human breast tumors was evaluated at the level of the single cell to better understand how breast tumor cells regulate expression in response to oxidative stress. Compared to normal breast ductal cells, the level of 8-oxo-dGTPase expression in the breast tumor cells increased from non-detectable levels in normal breast to expression in 30–85% of the tumor cells in individual tumors. There was no significant association between 8-oxo-dGTPase expression and tumor grade and metastatic malignancy. The upregulation of 8-oxo-dGTPase was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53, Ki-67 and c-erbB-2, which are genes involved in cell cycle regulation and tumor growth. The elevated expression of 8-oxo-dGTPase in human breast ductal carcinoma cells appears to be a general characteristic of breast tumors and may provide the tumor cell with a cellular defense mechanism to prevent the incorporation of 8-hydroxy-deoxyguanosine during DNA replication.

Reactivity of antibodies against 10-amino acid residue and pro-domain of stromelysin-3

Stromelysin-3 (ST3) has two highly conserved domains in the pro-domain. In particular, an unusual 10-amino acid residue sandwiched between the pro-domain and the catalytic domain of ST3 exists in ST3 but not in other matrix metalloproteinases (MMPs). To specifically detect ST3 expression in human tumors, we have made two kinds of ST3-specific polyclonal antibodies. One was raised against the synthetic 10-amino acid residue ( 88GLSARNRQKR 97) specific to ST3, and the other against recombinant ST3 pro-domain ( 62APATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKR 97) containing the decapeptide and PRCGVPD sequence obtained by expression in Escherichia coli. Two protein species, 59 kDa and 45 kDa which were consistent with those expected for pro-ST3 and the mature form of ST3, were specifically detected in 100-fold concentrated conditioned media of fetal lung fibroblast by Western blot analysis. Immunohistochemical staining indicated that in infiltrating ductal breast carcinoma and squamous cell carcinoma of the uterine cervix, reactivity of those antibodies was found not only in fibroblastic cells surrounding cancer cells but also in neoplastic cells. However, reactivity of two ST3 antibodies was inhibited by excess of the synthetic peptide (10-amino acid residue) not only in fibroblastic cells but also in neoplastic cells. These findings suggest that antibodies against the ST3 specific region may cross react with the recently known membrane type-metalloproteinase (MT-MMP), which have RXKR sequences between the pro- and catalytic domain.